Cultured PC12 cells provide a model well suited for the study of events associated with peptide factor-stimulated neuronal differentiation. PC12 cells possess high affinity receptors for/and respond to the neurotrophin, nerve growth factor (NGF). The neural differentiative biochemical signals elicited by NGF are primarily mediated through protein kinase cascades initiated by ligand-induced activation of the high affinity, NGF receptor tyrosine kinase p140trkA. In addition to p140trkA, PC12 cells express receptors for numerous other peptides including the mitogen, epidermal growth factor (EGF) and the neuroactive peptide, pituitary adenylate cyclase activating protein (PACAP). Analogous to p140trkA, the receptor for EGF is a tyrosine kinase. Upon addition of EGF to PC12 cells, virtually all the biochemical events that immediately follow receptor activation, particularly protein tyrosine phosphorylation of key signalling substrates, are identical to those observed for NGF. Addition of PACAP to PC12 cells results in activation of adenylate cyclase, subsequent generation of cyclic AMP and subsequent induction of cyclic AMP-dependent protein kinase A (PKA) activity. Addition of PACAP to PC12 cells induces sprouting of neuritic processes qualitatively dissimilar to the more robust, complexely arborized neurites elicited by NGF. Neither EGF nor PACAP, alone, elicits a complete morphologic neuronal differentiative response when added to PC12 cells, however, when the two peptides are concomitantly added, PC12 cells undergo morphololgic changes analogous to those observed for NGF. Pretreatment with the NGF-receptor-selective antagonist, K252a, completely abrogates responses to NGF, but is without effect on changes induced by co-administration of PACAP38 and EGF. Our studies have focused particular attention on ligand-mediated activation of the Ras/Raf/MEK/MAPK signal transduction pathway that has been demonstrated to play a critical role in NGF signaling. Time course analyis indicates that combination of EGF and PACAP results in MAPK activation kinetics that are comparable to those observed for NGF, whereas, treatment with either peptide alone fails to mimic NGF. Though the effects of PACAP in PC12 cells have been linked to PKA activation through stimulation of adenylyl cyclase, pretreatment with the PKA-selective antagonist, H-89, failed to inhibit stimulation of MAPK by any of the peptide combinations tested. However, pharmacologic inhibition of protein kinase C blocks activation of MAPK and prevents the outgrowth of neuritic processes, a hallmark feature of morphologic neuronal differentia-tion. Furthermore, unlike the situation for NGF, the effects of PACAP38 on PC12 cell morphology are independent of the activities of both Ras and Src. These findings are currently in press in Molecular Pharmacology (September, 1998).